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Cold Spring Harbor Laboratory Press
Protocol
Density Gradient Ultracentrifugation to Isolate Endogenous Protein
Complexes after Affinity Capture
Javier Fernandez-Martinez, John LaCava, and Michael P. Rout1
Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, New York 10065
This protocol describes the isolation of native protein complexes by density gradient ultracentrifugation. The outcome of an affinity capture and native elution experiment is generally a mixture of (1) the
complex(es) associated with the protein of interest under the specific conditions of capture, (2)
fragments of the complex generated by degradation or disassembly during the purification procedure,
and (3) the protease or reagent used to natively elute the sample. To separate these components and
isolate a homogeneous complex, an additional step of purification is required. Rate-zonal density
gradient ultracentrifugation is a reliable and powerful technique for separating particles based on
their hydrodynamic volume. The density gradient is generated by mixing low- and high-density
solutions of a suitable low-molecular-weight inert solute (e.g., sucrose or glycerol). The gradient is
formed in a solvent that could be any of the solvents used for the affinity capture and native elution and
should help to preserve the structure and activity of the assembly.
MATERIALS
It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental
Health and Safety Office for proper handling of equipment and hazardous material used in this protocol.
Reagents
Buffer for preparing gradients
As a general rule, the buffer used to prepare the gradients should be the same composition as the one used to
purify and stabilize the protein complex (i.e., the one used during the native elution during Protocol: Native
Elution of Yeast Protein Complexes Obtained by Affinity Capture [LaCava et al. 2015]). However, the buffer
composition can be modified depending on the downstream sample processing requirements, bearing in mind
that the stability and activity of the complex in the new composition should be tested beforehand.
Natively eluted yeast protein complexes prepared by either method described in Protocol: Native
Elution of Yeast Protein Complexes Obtained by Affinity Capture (LaCava et al. 2015)
Protease inhibitor cocktail (e.g., Sigma-Aldrich P8340)
Sucrose (66%, w/w) or glycerol
Prepare a solution of 66% (w/w) sucrose by dissolving 1710 g of sucrose (molecular biology grade) in 900 mL of
high-performance liquid chromatography (HPLC)-grade water. Double-check the concentration using a refractometer and correct if necessary by adding sucrose or water. This solution can be stored at 4˚C indefinitely
because the low available water in the solution prevents bacterial growth.
As an alternative to sucrose, glycerol is a solute very commonly used to generate density gradients.
1
Correspondence: [email protected]
© 2016 Cold Spring Harbor Laboratory Press
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Centrifugation to Purify Affinity-Captured Protein Complexes
Equipment
Filter (Millex-GP Syringe Filter Unit [0.22-µm] from EMD Millipore)
Gradient Master (BioComp Instruments)
Piston Gradient Fractionator (BioComp Instruments) (optional; see Step 10)
Polyclear centrifuge tubes (13 × 51-mm) (Seton Scientific)
When purchased from BioComp Instruments, Inc. (cat. no. 151-513), the tubes are tested for tolerance and
compatibility with piston fractionation.
Refractometer (Bausch & Lomb)
SW 55 Ti Rotor (Beckman Coulter)
Ultracentrifuge (Beckman Coulter)
METHOD
Gradient Preparation
Prepare two gradients by following the instructions in the BioComp Gradient Master operator’s manual, which are
briefly summarized here. The small gradient volumes (!5 mL each) help to maintain the concentration of the purified
complex within a reasonable range, as usually the amount of purified native protein assemblies is very limited.
1. Prepare 10 mL each of 5% and 20% (w/w) sucrose solutions in a buffer with the same composition as the one used during the native elution. See Griffith (1986, p. 49) for a chart indicating the
appropriate dilutions of a 66% (w/w) sucrose solution to prepare a 5% solution (1/20 dilution)
and a 20% solution (1/4.1 dilution). Filter the solutions through a 0.22-μm filter. Verify the final
concentration using a refractometer.
The concentrations of the gradient should be adjusted to the size of the protein complex that needs to be
isolated. 5%–20% (w/w) sucrose works well for complexes of 200–800 kDa. For larger complexes, a 10%–
40% (w/w) sucrose gradient would be more suitable. If the gradient is prepared using glycerol, the most
common gradient used for separation of protein complexes is 10%–30% (v/v).
2. Add protease inhibitor cocktail to each sucrose solution to give a final concentration of 0.001×,
and mix carefully by rocking to avoid the formation of air bubbles. Add other kinds of chemical
compounds depending on the enzymatic activities that need to be inhibited or preserved.
3. Place two polyclear centrifuge tubes into the SW 50/SW 55 marker block and, using a marker,
draw a fine line following the top of the block. Fill each tube with 5% (w/w) sucrose solution until
the liquid reaches !2 mm above the half-full mark. Fill a 10-mL syringe with the 20% (w/w)
sucrose solution and attach a blunt-end steel cannula to its tip. Being careful to avoid air bubbles
and not to disturb the interface, insert the cannula quickly to the bottom of the tube and slowly
layer the 20% solution until the interface formed with the 5% reaches the half-full mark. Quickly
withdraw the cannula and cap the tubes using the short rubber caps.
4. Place the tubes in the tube holder and use the Gradient Master to rotate and mix the solutions as
outlined in the operator’s manual to generate a linear gradient.
5. Take the formed gradients and let them cool down for 45 min to 1 h at 4˚C. At this time, ensure
that the rotor is also precooled at 4˚C.
Centrifugation
The gradients are stable for up to 3 h at 4˚C.
During this series of steps, avoid heating the gradients by working fast or in a cold room.
6. Carefully remove the caps from the gradient tubes, and slowly load between 50 and 200 µL
containing 10–100 pmol of natively eluted yeast protein complexes (from Protocol: Native
Elution of Yeast Protein Complexes Obtained by Affinity Capture [LaCava et al. 2015]) on
top of one of the gradients, trying not to disturb it.
Although these ranges are desirable, lower amounts can be successfully analyzed. We recommend using
gel-loading tips for this task.
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J. Fernandez-Martinez et al.
7. Use the other gradient to balance the rotor; add a volume of water or buffer equal to the volume of
sample so as to keep them balanced.
Alternatively, to analyze several samples, distribute the samples across multiple gradients.
8. Balance the tubes before loading them into the swing-out rotor. Check that the gradients are of
comparable weight using a balance, keeping them within ±100 mg of one another by adding
solution as needed.
9. Centrifuge at 59,000–237,000g in an SW 55 Ti rotor and Beckman L-80 ultracentrifuge for 6–20 h
at 4˚C.
These centrifugation conditions are commonly used, but the time and speed of centrifugation must be
determined empirically for each protein assembly because their sedimentation rate depends not only on
their size, but also on their shape. Ideally, the complex should move about two-thirds of the way down the
gradient to obtain good separation.
10. After centrifugation, fractionate the gradients at 4˚C by one of the following methods.
• Use a BioComp Piston Gradient Fractionator to collect fractions in 2-mm increments (i.e.,
!20 fractions of !225 µL each).
•
Manually fractionate by unloading from the top using a pipette or from the bottom of the tube
using a capillary and a peristaltic pump. Collect 12 fractions of 410 µL each or 24 fractions of
205 µL each, depending on how precisely the peak of the complex needs to be determined.
For assays requiring native complexes (e.g., assays of enzymatic activity or protein complex structure),
analyze the fractions immediately. If this is not possible, store for short periods (no more than a few
hours) at 4˚C, or flash-freeze in liquid nitrogen and store for days to months at –80˚C. For assays that do
not require native complexes (e.g., sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDSPAGE] analysis), store the fractions at –20˚C until analysis.
Analysis of Fractions
11. Analyze an aliquot of each fraction by SDS-PAGE protein staining (e.g., Coomassie blue, silver, or
SYPRO Ruby) to visualize the components and localize the complex of interest.
In the event that the final sample concentration in the fractions is insufficient for visualization upon direct
loading of an aliquot, the fractions can be effectively precipitated with methanol/chloroform (Wessel and
Flügge 1984).
Data from an experiment in which a native protein complex was purified by affinity capture, released by
PreScission protease cleavage, and enriched by density gradient ultracentrifugation is presented in Figure 1.
12. Characterize the purified native complexes as desired.
Natively eluted
complex (input)
Protease:
Nup84p-SpA
–
5%
20% sucrose
MW (kDa)
+
212
5%
Nup133p
158
Nup133p
Nup120p
116
97
Nup120p
Nup84p/85p/145c
Nup84p/85p/145c
Ultracentrifugation
66
55
Sucrose
42
Seh1p
Seh1p
35
Sec13p
27
Sec13p
Protease
20%
Protease
20
In
1
2
3
4
5
6
7
8
9
10 11 12
FIGURE 1. The endogenous heptameric Nup84-complex purified by affinity capture, released by PreScission protease
cleavage, and enriched by fractionation across a 5%–20% (w/w) sucrose density gradient.
626
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Centrifugation to Purify Affinity-Captured Protein Complexes
Mass spectrometry is the ideal method to identify the proteins present in each fraction. Depending on the
downstream application, the sucrose or glycerol used in the gradient may need to be removed by dialysis,
gel filtration, or ultrafiltration. For examples of downstream structural and functional characterization using
protein complexes purified with this procedure, see Erickson (2009) and Luhrmann and Stark (2009).
RELATED INFORMATION
For further information regarding the density gradient ultracentrifugation procedure, consult Griffith
(1986) and Erickson (2009).
REFERENCES
Erickson HP. 2009. Size and shape of protein molecules at the nanometer
level determined by sedimentation, gel filtration, and electron microscopy. Biol Proced Online 11: 32–51.
Griffith OM. 1986. Techniques of preparative, zonal, and continuous flow
ultracentrifugation. Beckman, Palo Alto, CA.
LaCava J, Fernandez-Martinez J, Rout MP. 2015. Native elution of yeast
protein complexes obtained by affinity capture. Cold Spring Harb
Protoc doi: 10.1101/pdb.prot087940.
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot087957
Luhrmann R, Stark H. 2009. Structural mapping of spliceosomes by electron
microscopy. Curr Opin Struct Biol 19: 96–102.
Wessel D, Flügge UI. 1984. A method for the quantitative recovery of protein
in dilute solution in the presence of detergents and lipids. Anal Biochem
138: 141–143.
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